1. Field of the Invention
The present invention relates generally to the fields of diagnostic and screening tests. More particularly, it concerns improved methods for the detection of early anti-HIV antibody in a sample, as well as early diagnostic tests to detect human immunodeficiency virus exposure or infection in infants. An additional aspect of the invention concerns methods for detecting HIV in idiopathic chronic lymphopenia patients. The invention also relates to the field of recombinant proteins, as particular recombinant proteins and recombinant protein composite preparations of several human immunodeficiency virus strains are disclosed. The invention also relates to the field of commercial diagnostic and prognostic assay plates and kits, as assay plates designed to detect an early anti-HIV antibody that include the described recombinant human immunodeficiency virus antigen or preparations of human immunodeficiency virus infected cell isolates are described.
2. Background of the Invention
Patients infected with human immunodeficiency virus (HIV) are known to eventually mount a humoral immune response to the virus. The production of anti-HIV antibody is one marker used to detect this response. In some studies, anti-HIV antibody has been reported to be more reliable for diagnosis than either HIV culture or HIV antigen detection in patient samples1,2. Consequently, anti-HIV antibody detection tests are the most common method of diagnosis of infection. Both EIA and Western blot assays are currently used in the detection of anti-HIV antibody.
Unfortunately, a degree of unreliability continues to exist with the use of conventional anti-HIV antibody screening methods, such as by conventional EIAs. Thus, a further confirmatory test, such as a Western Blot (WB) or fixed-cell immunofluorescence assay, have become recommended additional testing procedures.
Despite these and other additional precautionary testing measures, a number of studies report the existence of a seemingly silent period of HIV infection during which antibody to the virus is not detectable even after exhaustive testing. This period reportedly extends from the point of infection to the time infection is detectable through conventional sero-conversion assays. This silent period has been reported to persist anywhere from a few months to as much as two and one-half years before infection is detectable by conventional EIAs and Western blot assays.
While not always successful, culturing of peripheral blood lymphocytes to amplify HIV does provide for detection of the virus when anti-HIV antibody cannot be detected by conventional EIA or WB. However, several recent studies using PCR-based HIV detection methods continue to report the existence of PCR(+)positive, sero(xe2x88x92)negative cases in high-risk populations10-17. Nevertheless, PCR usually does detect infection before conventional sero-conversion methods, with the aforedescribed period of silent infection being reduced by approximately one month, at least in some cases18.
The rate of HIV transmission in negatively tested blood, using conventional testing methods, continues to persist at a relatively constant rate25. For example, HIV-1 transmission from seemingly xe2x80x9cseronegativexe2x80x9d blood using EIA conventional methods, continue to be reported21-23. Donated organs also constitute a source of HIV disease transmission, with HIV infection being diagnosed in recipients of organs from individuals whom, again, test HIV seronegative by conventional assays24.
Retrospective studies have reported that early donor education and self-exclusion measures has reduced the rate of disease transmission26. However, such exclusion methods together with antibody testing, while hopefully reducing the probability of at least some false negative results27, provides only a partial and imperfect solution to the problem in at least a small subset of reported HIV cases.
Some studies report the presence of HIV specific T-cells in high risk individuals testing negative with conventional EIA, WB, and PCR based detection techniques28,29. Other reports have identified the existence of B-cells which produce HIV-specific antibodies in vitro that are present in EIA-negative, WB-negative, high-risk subjects30. While these approaches present possible alternatives, for testing, they are relatively complex and difficult procedures, and are thus impractical for large-scale clinical screening. The expense and time associated with this type of testing again leaves a need in the medical arts for a reliable and practical HIV screening and detection approach.
Early HIV infection of infants is a particularly troublesome problem. Current technology renders it difficult to diagnose whether an infant less than 18 months of age is infected, absent development of overt clinical symptoms. Conventional HIV serological tests for anti-HIV antibody are inadequate for detecting infection in an infant because the antibody detected is not necessarily that of the infant, but is that of the HIV-positive mother. This maternally derived antibody typically persists for up to 21 months in the infants system34.
Neither IgA or IgM antibody cross the placenta. Hence, studies in children have emphasized the detection of IgA and IgM as indicators of infant HIV infection. In one study, both HIV-specific IgA and IgM were found in infants up to 12 months of age born to sero-positive mothers, with twice as many samples yielding IgA anti-HIV compared to IgM (66% vs. 33%)35 using conventional screening assays (WB, EIA).
Currently, approximately 50% of infected infants can be identified at birth, approximately 90% by 3 months of age, and almost all by 6 months of age using combination HIV culture, PCR, IgA antibody tests, and p24 antigen tests38. However, the fact that HIV can be detected in only one-half of infected infants at the time of birth again points to the continued need for improved early HIV detection in infants.
The present invention, in a general and overall sense, concerns recombinant HIV envelope proteins and peptides, and early anti-HIV antibody immunoreactive fragments thereof, that are capable of immunologically binding to early anti-HIV antibodies.
As used in the description of the present invention, early anti-HIV antibodies are defined as the first anti-human immunodeficiency virus antibodies that are induced in a human infected with the HIV virus, these antibodies being capable of recognizing conformational epitopes of HIV gp160 antigen and which are not detectable by current EIA or Western Blot assay using HIV gp160 target antigen that has not have retained conformational epitopes.
The invention further provides for an improved HIV detection and screening method by allowing for the identification of early anti-HIV antibody. These early anti-HIV antibodies previously went undetected using conventional assays because the target antigens historically employed in these assays lacked sufficiently preserved conformational epitopes necessary for early antibody recognition.
The compositions and assays of the present invention comprise improved HIV target antigens that include the conformational epitopes of an HIV envelope protein, specifically the HIV gp160. The early anti-HIV antibodies detectable using the described antigen do not recognize primary sequence epitopes, and hence they go undetected with conventional serological tests that employ at least partially denatured HIV target antigen (EIA and Western blot).
Because PCR-based assays provide little improvement over conventional EIA and Western Blot serological-based assays for detecting HIV, it is expected that the presently described EIA""s will also provide an improved method over PCR based detection and screening methods.
Recombinant Proteins and Peptides of Human Immunodeficiency Virus
In some embodiments of the invention, a recombinant protein comprising a recombinant human immunodeficiency virus envelope protein capable of immunologically binding an early anti-HIV antibody is provided. For example, these recombinant proteins in particular embodiments may be further defined as having a molecular weight of about 160,000 Daltons as determined by SDS/PAGE. In these particular forms, the recombinant HIV protein is an HIV envelope protein, HIV gp160.
The recombinant HIV envelope proteins of the invention may further be described by reference to the process by which they are prepared. For example, in one embodiment, the process comprises preparing a nucleic acid sequence or a restriction fragment of the human immunodeficiency virus nucleic acid sequence that encodes a human immunodeficiency virus envelope protein, such as gp160; inserting the restriction fragment into a vector capable of transfecting a eukaryotic cell to provide a recombinant vector; transfecting eukaryotic cell capable of expressing the human immunodeficiency virus envelope protein capable of binding an early anti-HIV antibody to provide a recombinant eukaryotic cell; culturing the recombinant mammalian cell under conditions suitable for expression of the recombinant HIV protein; and collecting recombinant HIV envelope protein capable of binding early anti-HIV antibody.
An example of a mammalian cell capable of expressing the HIV envelope protein nucleic acid sequence as described here is the CEM cell line. Such CEM cell lines are available through the American Type Culture Collection (ATCC). An example of the vector capable of transfecting a mammalian cell is a retroviral vector, such as pLNSX. Other carriers, such as an adenovirus or a plasmid, may also be used to transfer the HIV envelope protein encoding nucleic acid sequence. The recombinant retroviral vector in a particular embodiment of the process is the pLNSX-env.
In other embodiments of the recombinant human immunodeficiency virus protein, the protein is described as comprising a recombinant gp160 human immunodeficiency virus glycoprotein or fragments of the gp160 protein, such as the gp120 or gp41 fragment of the gp160 protein.
The recombinant HIV envelope proteins and peptides of the invention are further described in some embodiments as comprising recombinant proteins and peptides prepared from substantially purified and non-denatured lysates of mammalian cells transfected with vectors expressing recombinant human immunodeficiency virus, as well as recombinant proteins/peptides comprising the substantially purified and non-denatured expression products of these transfected mammalian cells.
In yet other embodiments, the recombinant protein/peptide preparations comprise a composite of recombinant human immunodeficiency virus gp160 proteins, the composite comprising recombinant gp160 protein from more than one human immunodeficiency virus strain.
Mammalian cell lines that are transfected with retrovirus expression vectors carrying the gp160 encoding-human immunodeficiency virus gene have been prepared with gp160-encoding nucleic acid fragments obtained from several different strains of the human immunodeficiency virus obtained from HIV-infected patients. For example, recombinant HIV envelope protein has been prepared from HIV213 virus strain.
In a particular embodiment, the protein/peptide gp160 composite preparation comprises the recombinant gp160 protein expression product of at least three different HIV strains found to be immunoreactive with the early HIV antibody detected in a representative number of serum samples determined to be seronegative by conventional EIA and Western Blot techniques. By way of example, three such HIV strains are HIV213, HIVC and HIVAC-1. These HIV strains have been deposited with the American Type Culture Collection depository (12301 Park Lawn Drive, Rockville, Md. 20852). The deposit information is as follows:
HIV213xe2x80x94ATCC VR 2247
HIVAC-1xe2x80x94ATCC VR 2246
HIVTP-1xe2x80x94ATCC VR 2245
Target antigen may be prepared from either the expression product obtained from eukaryotic cells transfected with retroviral or other vectors carrying these gp160 encoding nucleic acid sequences or fragments thereof, or from substantially nondenatured lysates of such transfected eukaryotic cells.
As used in the description of the present invention in the description of the human immunodeficiency virus recombinant protein/peptide preparations and cell lysates, the phrase xe2x80x9csubstantially non-denaturedxe2x80x9d in used to define a protein having a preserved configurational integrity of the human immunodeficiency virus envelope gp160 protein or a portion thereof sufficient to bind early anti-HIV antibody. Conformation of the protein/peptide used as target antigen is important in providing this early antibody recognition.
The present inventor provides here procedures that preserve sufficient conformational integrity of the protein/peptide to allow early anti-HIV binding recognition. It is anticipated that given the disclosure here, other similar protein/peptide preparation processes may be devised that result in useful target antigen compositions for early anti-human immunodeficiency virus screening. All such modified procedures, insofar as they represent minor or insignificant modification of the procedures and specific materials described herein, are therefore intended by the inventor to be embraced within the scope of the present invention.
While a number of different HIV strains were examined by the present inventor, other HIV viral strains not specifically mentioned or examined here may also be employed in the preparation of the various HIV proteins/peptides of the invention. It is expected that other HIV viral strains may be used to provide the defined substantially preserved conformational epitopically intact HIV proteins capable of early anti-HIV antibody recognition. The particularly noted HIV strains used to create recombinant protein/peptide target antigen were selected based on an observed activity to bind early anti-HIV antibody in human patient serum or plasma samples determined to be seronegative by conventional antibody testing procedures. Hence, additional such representative strains may be identified and selected using the procedures outlined herein, and subsequently processed again according to the procedures described in detail here in providing recombinant HIV antigen also useful in screening and diagnosing early anti-HIV antibody and HIV infection in a patient sample.
In a particular embodiment, the recombinant protein/peptide of the invention comprises a sufficiently conformationally intact HIV envelope protein capable of immunologically binding an early anti-HIV antibody, said antigen being isolatable as an expression product from a mammalian or other eukaryotic cell transfected with an expression vector having a sequence encoding a gp160 HIV envelope protein. The HIV gp160 envelope protein, by way of example, may be that expressed by a eukaryotic cell infected with a viral strain HIVC, HIV213, or HIVAC-1. It is also expected that other HIV isolate strains that are capable of expressing the gp160 antigen, or instead the gp41 or gp120 HIV envelope antigen, may be used in conjunction with the present invention as well.
Purified Preparations of HIV Envelope Proteins
Further aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of the described recombinant HIV envelope protein preparations. The term xe2x80x9cpurified envelope proteinxe2x80x9d as used herein, is intended to refer to a protein composition that is isolatable from eukaryotic cells, either in the form of a solubilized population of HIV infected cells or as a protein from recombinant-expressing cells. These purified envelope proteins are further defined as essentially non-denatured and capable of binding with early anti-HIV antibody. The HIV envelope protein is purified to any degree relative to its naturally-obtainable state. In some embodiments, the purified antigen state is relative to purity of the recombinant antigen as it exists in a whole mammalian cell lysate. A purified HIV envelope protein therefore also refers to a recombinant envelope protein, free from the environment in which it may naturally occur in intact recombinant-expressing mammalian cells.
Generally, xe2x80x9cpurifiedxe2x80x9d will refer to an HIV envelope protein composition wherein various non-HIV envelope components, such as other cell components, have been removed, and which composition substantially retains its antigenicity and/or capacity to interact with early anti-HIV antibody. Where the term xe2x80x9csubstantially purifiedxe2x80x9d is used, this will refer to a composition in which the human immunodeficiency virus envelope protein forms the major component of the composition, such as constituting about 50%, about 60%, about 80% or about 95% of the protein in the composition or more.
Various methods for quantifying the degree of purification of the HIV protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the activity of the preparation for detecting early anti-HIV antibody by assessing the number of different polypeptides within a fraction by SDS/PAGE analysis. In some embodiments, the recombinant HIV protein is to be purified to homogeneity expressed as an about 80% purity from a crude cell lysate so as to be identifiable in a single band of an SDS page gel stained with comassie blue. SDS page gel analysis of a protein preparation is well known to those of ordinary skill in the art.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. A specific example presented herein is the purification of recombinant gp/60 expressed mammalian cells which are solubilized using affinity binding to anti-gp41 monoclonal antibody bound to wells of plastic EIA plates.
Some of the recombinant HIV protein preparations from transfected mammalian cell lysate of the present invention have a purity of 80%. This number represents a ratio of, for example, 8 gp160 HIV antigen molecules to two non-gp160 molecules. Preparations of lower or higher purity are also useful in detecting the early anti-HIV antibody.
The preferred purification method disclosed hereinbelow contains several steps and represents the best mode presently known by the inventors to prepare a substantially purified recombinant HIV envelope protein and still maintain its native conformation. This method is currently preferred as it results in the substantial purification of the recombinant HIV protein, as assessed by SDS-PAGE determinations and ability to bind to early antibodies, while simultaneously coating EIA plates. This preferred mode of recombinant protein purification from HIV transfected mammalian cells involves the execution of certain steps in the order described hereinbelow. However, as is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified recombinant HIV envelope protein, such as the recombinant gp160 HIV envelope protein.
As mentioned above, although preferred for use in certain embodiments, there is no general requirement that the HIV transfected mammalian cell lysate always be provided in their most purified state. Indeed, it is contemplated that less substantially purified cell lysates, which are nonetheless enriched in recombinant HIV envelope protein, such as gp160 relative to the natural state, will have utility in certain embodiments. These include, for example, the detection of early anti-HIV antibody. Partially purified HIV transfected mammalian cell lysate fractions for use in such embodiments may be obtained by subjecting an HIV transfected mammalian cell lysate extract to one or a combination of the steps described herein, the HIV recombinant envelope preparations may also be obtained as a secreted product from cells carrying an HIV envelope antigen (e.g. gp160) encoding nucleic acid sequence.
Recombinant Vectors
The recombinant vectors of the invention may comprise any vehicle capable of transfecting a eukaryotic cell. By way of example, such vehicles include retrovirus vectors and adenovirus vectors. Plasmids may also be used to transfer HIV nucleic acid fragments encoding the HIV envelope proteins of the invention. In some embodiments, the retroviral vector comprises a nucleic acid sequence encoding a human immunodeficiency virus envelope protein, such as the gp160, or that encodes an early anti-HIV antibody immunoreactive fragment of the human immunodeficiency virus envelope protein.
In particular embodiments, the nucleic acid sequence encoding the human immunodeficiency virus envelope protein comprises an SalI-XhoI restriction fragment of a human immunodeficiency virus nucleic acid sequence. By way of example, the inventor has prepared SalI-XhoI restriction fragments of several human immunodeficiency viruses, particularly HIV strains C, 213 and AC-1, and blunt-end ligated the fragment into a retroviral vector, such as the pLNSX retroviral vector.
Mammalian Cell Lines
The present invention in another aspect provide recombinant mammalian cells that express the recombinant human immunodeficiency virus envelope proteins and peptides. Any variety of mammalian cells or cell lines may be used, as long as they are capable of expressing the recombinant HIV protein capable of immunologically detecting the early anti-HIV antibody. By way of example, a cell line that may be used is a CEM human cell line, particularly described as CEM human T-cells.
Methods for Preparing Recombinant HIV Envelope Protein and Peptide
Methods for preparing the recombinant HIV envelope are also provided. In one particular embodiment, the method comprises transfecting a mammalian cell with a retroviral vector genetically engineered to include a sequence encoding a human immunodeficiency viral envelope protein. These transfected mammalian cells are then subject to a screening process, such as by antibiotic resistance to G418 for example, and clones selected that express the highest amount of the human immunodeficiency virus envelope protein. By way of example, clones expressing the highest amounts of HIV envelope protein could be selected using an anti-HIV gp160 monoclonal antibody (e.g., DZ33, Medarex, Inc.).
One particular clone expressing the HIV envelope protein gp160 isolated by the present inventors is Clone 7. Clone 7 was obtained by transfection of a CEM cell line with LNSX retroviral vector containing the env gene of HIV213 strain. This clone 7 was examined by flow cytometry, and shown to express high levels of the recombinant HIV gp160 protein (FIG. 5).
Other clones that express a fusion protein of gp160 obtained from more than one of the HIV strains may also be obtained. Such would be achieved, for example, by constructing a retroviral vector that included a gp160 encoding fragment of nucleic acid obtained from the desired HIV isolates, and then transfecting a mammalian cell line with said construct.
Any of the herein described recombinant HIV antigens may be used alone or in combination as a target antigen for the detection of early anti-HIV antibody. Combinations of these recombinant antigens from the noted viral strains have proven to be particularly efficacious for detecting the early anti-HIV antibody in the widest range of patient samples examined to date.
Methods/Processes of Producing Recombinant HIV Envelope Protein/Peptide
The invention also provides for methods/processes of producing a recombinant HIV antigen. In one embodiment, the method comprises obtaining a nucleic acid fragment encoding a human immunodeficiency virus gp160 envelope protein; ligating the nucleic acid fragment into a vector to provide a recombinant vector; transfecting a mammalian cell with the recombinant vector to provide a transfected mammalian cell; and collecting recombinant gp160 protein. The recombinant gp160 protein may be collected by virtue of solubilizing the infected mammalian cell in a solubilizing agent that does not destroy the early anti-HIV antibody detecting capability of the antigen, such as digitonin, and then removing the cellular components in the solubilized preparation. Alternatively, the recombinant HIV antigen may be collected as a secreted product from recombinant eukaryotic cells, such as from a yeast cell that has been genetically engineered such that it secretes the gp160 HIV envelope protein.
A method that was used to collect recombinant gp160 that was used as target antigen to detect early anti-HIV antibody in a sample was through the use of 1.0% digitonin as a solubilizing agent on CEM cells infected with HIV. This procedure is described at Example 3. The recombinant HIV gp160 antigen was captured onto an ELISA plate by pouring the whole recombinant cell lysate into wells of the plate, which will be coated with mouse anti-gp41 muAB. The plate was then washed to remove cellular components and other antigens. In this fashion, the desired HIV gp160 antigen was isolated on the plate.
In a particular embodiment, the method or preparing a recombinant gp160 envelope protein comprises: obtaining a nucleic acid fragment encoding a gp160 HIV envelope protein; inserting said fragment into a vector capable of transfecting a mammalian cell; and transfecting a mammalian cell capable of expressing the gp160 envelope protein with said vector. The method may include the further step of collecting the recombinant gp160 protein.
Assay Plates
In some embodiments, the wells of the assay plates may first be coated with an anti-gp41 or anti-gp160 antibody. This would immobilize HIV gp160 antigen to the plastic in the presence of a mild solubilizing buffer, such as from about 0.1% to about 10% digitonin (particularly about 1% digitonin). Such an approach is particularly efficacious in preparing assay plates with wells made of plastic.
The assay plates in other embodiments of the invention comprise a multiplicity of microtiter wells, and in some embodiments, polystyrene microtiter wells. These wells would be coated with about 500 ng/well of the recombinant HIV envelope protein, or recombinant HIV antigen or HIV-infected whole cells or cell lysates thereof.
Early Anti-HIV Antibody Detection Assays
In yet another embodiment, the invention provides for improved anti-HIV antibody detection assays using the aforedescribed native or recombinant HIV proteins/peptides as target antigen. These improved assays, particularly ELISA sandwich-type assays, provide for the detection of early anti-HIV antibodies in sandwich-type assays, provide for detection of early anti-HIV antibody in samples that test anti-HIV antibody negative using conventional EIA and WB techniques.
The format of the EIA may be described as employing plates that are directly coated with the HIV antigen (the antigen being either recombinant HIV envelope protein expressed from HIV-transfected mammalian cells, or a lysate of substantially non-denatured, solubilized HIV-infected mammalian cells that express the HIV envelope protein), or plates that are designed to function as an antibody capture sandwich assay.
Immunoassays
As noted, it is proposed that the recombinant gp160 of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of anti-gp160 conformational epitope-reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs) known to the art. However, it will be readily appreciated that the utility of the gp160 preparations described herein are not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays or procedures.
In some embodiments of the ELISA assay, native gp160 or appropriate peptides incorporating gp160 antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one will desire to bind or coat a nonspecific protein such as bovine serum albumin (BSA), casein, solutions of milk powder, gelatin, PVP, superblock, or horse albumin onto the well that is known to be antigenically neutral with regard to the test antisera. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. Following an appropriate coating period (for example, 3 hours), the coated wells will be blocked with a suitable protein, such as bovine serum albumin (BSA), casein, solutions of milk powder, gelatin, PVP, superblock, or horse albumin, and rinsed several times (e.g., 4 or 5 times) with a suitable buffer, such as PBS. The wells of the plates may then be allowed to dry, or may instead be used while they are still wet.
After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 1 to 4 hours, at temperatures preferably on the order of 20xc2x0 to 25xc2x0 C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human IgG, IgM or IgA. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease, alkaline phosphatase, or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2xe2x80x2-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
In each of the microtiter wells will be placed about 10 xcexcl of the test patient sample along with about 90 xcexcl of reaction buffer (e.g., PBS with about 1% digitonin or other mild protein solubilizing agent). Control wells of the ELISA plate will include normal sera (human sera without early anti-HIV antibody), early anti-HIV antibody collected from HIV patient subjects who had not sero-converted as assessed using Western blot, and late anti-HIV antibody obtained from patients that have seroconverted using conventional anti-HIV antibody detection techniques.
Early HIV Infection Detection in Infants
In a particular embodiment, the invention provides for a method for detecting early IgM or IgA anti-HIV antibody in a young child. These methods are improved over existing techniques, as they provide for the earlier detection of HIV infection in infants under the age of 12 months.
Early Anti-HIV Antibody Detection Kits
In yet another aspect of the invention, a kit is envisioned for early anti-HIV antibody detection. In some embodiments, the present invention contemplates a diagnostic kit for detecting early anti-HIV antibodies and human immunodeficiency virus infection. The kit comprises of reagents capable of detecting the early anti-HIV antibody immunoreactive with the native or recombinant HIV antigen described here.
In some embodiments, the kit may also comprise a container means comprising a secondary antibody capable of detecting the early anti-HIV antibody which is immunoreactive with the recombinant HIV envelope antigen.
The HIV antigen reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, plastic beads or plates, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent. In yet other embodiments, the kit may further comprise a container means comprising an appropriate solvent.
In some embodiments, the kit comprises a container means that includes a volume of a second antibody, such as goat anti-human IgG or IgM conjugated with alkaline phosphatase or other anti-human Ig secondary antibody, and a second container means that includes a volume of a buffer comprising a non-denaturing solubilizing agent, such as about 1% digitonin.
The kit may in other embodiments further comprise a third container means that includes an appropriate substrate, such as PNPP for alkaline phosphatase, or 9-dianisidine for peroxidase. A fourth container means that includes an appropriate xe2x80x9cstopxe2x80x9d buffer, such as 0.5 m NaOH, may also be included with various embodiments of the kit.
The kit may further include an instruction sheet that outlines the procedural steps of the assay, and will follow substantially the same steps as the typical EIA format known to those of skill in the art.
Idiopathic Chronic Lymphopenia Screening
In yet another aspect of the invention, methods for screening samples for evidence of HIV in idiopathic chronic lymphopenia are provided. In some embodiments, the method comprises obtaining a biological fluid sample from a patient; exposing said sample to a native or recombinant human immunodeficiency virus envelope protein which is capable of binding early anti-HIV antibodies under conditions sufficient to allow immunocomplex formation between the labeled recombinant protein and any antibody present in the patient sample, so as to provide an incubation mixture; and identifying the presence of immunocomplex formation in the incubation mixture, via some label, wherein the presence of labeled immunocomplex formation provides a screen for HIV in idiopathic chronic lymphophenia.